The broad goal of this project is to develop fluorescent proteins with an 80-fold improvement in signal output (e.g. number of photons emitted before photobleaching). Over the last 10-15 years, fluorescent proteins have provided critical insights into the fundamental workings of the cell as they enable researchers to visualize protein movements, enzyme activities, gene expression, and to quantify important signaling molecules in real time in living cells. As a result, fluorescent proteins have truly revolutionized cell biology, shedding light on the basic biology of cellular function, while helping to elucidate what goes wrong in disease states. Substantial improvements in the signal output of fluorescent proteins are required for the next level: visualizing and monitoring of single molecules within individual living cells. Although there has been a concerted effort in the protein engineering field to enhance fluorescent protein properties, recent improvements have been incremental at best. We hypothesize that an 80-fold improvement in signal output can be obtained by explicitly engineering both the chromophore pocket and surface environment of fluorescent proteins. We propose to generate targeted libraries of these proteins, express the libraries in mammalian cells, and screen proteins for increased brightness, increased photostability, and decreased conversion to "dark states" using a novel microfluidic cell sorter that we recently implemented. Moreover we will conduct multi-parameter screens in order to identify mutations that enhance multiple photophysical properties (for example brightness and photostability) and combine synergistically to improve signal output substantially. This information is crucial as protein engineering efforts based on a single selection scheme typically optimize one property at the expense of another, leading to only modest gains in signal output. Our goal is to connect sequence diversity to functional diversity in order to provide insight into the molecular control of photophysical properties in the fluorescent proteins. This information will not only be used in future protein design efforts, but will help us define the maximum signal output obtainable from fluorescent proteins. The proposed research has 3 Specific Aims: (1) To increase signal 20-fold with multi-phase screen that probes dark-state relaxation rate;(2) To increase photostability of red fluorescent proteins by at least 7-fold with screen that probes photobleaching;(3) To identify combinations of mutations that yield synergistic effects on photophysical properties. This will enable us to combine enhancements identified in aim 1 and 2, with improvements in brightness to yield an 80- fold increase in signal output. We have chosen to focus on red fluorescent proteins because these have the greatest potential for impacting single molecule cellular imaging. The development of new fluorescent proteins with substantial increases in signal output will dramatically expand our ability to visualize and probe the inner workings of living cells. These imaging tools will impact public health by providing valuable insight into basic biology and mechanisms of disease progression.